Phospholipases C (EC 3.1.4.3) are a family of enzymes which hydrolyze the sn-3 phosphodiester bond in membrane phospholipids producing diacylglycerol and a phosphorylated polar head group. Mammalian phospholipase C (PLC) enzymes exhibit specificity for the polar head group which is hydrolyzed, i.e., phosphatidylcholine, phosphatidylinositol, etc. Recently, much interest has been generated in those PLC enzymes which selectively hydrolyze phosphoinositide lipids in response to receptor occupancy by agonist. Hydrolysis of phosphatidylinositol 4,5-bisphosphate generates two second messenger molecules; diacylglycerol, a co-factor required for activation of protein kinase C, and inositol 1,4,5-trisphosphate, a soluble second messenger molecule which promotes the release of intracellular nonmitochondrial stores of calcium (Berridge, Ann. Rev. Biochem., 56:159-193, 1987). The diacylglycerol released may be further metabolized to free arachidonic acid by sequential actions of diglycerol lipase and monoglycerol lipase. Thus, phospholipases C are not only important enzymes in the generation of second messenger molecules, but may serve an important role in making arachidonic acid available for eicosanoid biosynthesis in select tissues.
Mammalian tissues contain multiple distinct forms of phosphoinositide-specific PLC (Crooke and Bennett, Cell Calcium, 10:309-323, 1989; Rhee et al., Science, 244:546-550, 1989). It is proposed that each of the enzymes couple to distinct classes of cell surface receptors, i.e., PLC-.beta. couples to thromboxane A.sub.2, bradykinin, angiotensin and muscarinic receptors via G.sub.q .alpha. or G.sub.11 .alpha. (Shenker et al., J. Biol. Chem., 266:9309-9312 (1991); Gutowski et al., J. Biol. Chem., 266:20519-20524 (1991); Berstein et al., J. Biol. Chem., 267:8081-8088 (1992)), PLC-.gamma. couples to growth factor receptors, etc. (Aiyar et al., Biochem. J., 261:63-70, 1989; Crooke and Bennett, Cell Calcium, 10:309-323, 1989; Margolis et al., Cell, 57:1101-1107, 1989; Wahl et al., Proc. Natl. Acad. Sci. USA, 86:1568-1572, 1989). Alignment of sequences from all groups reveals that the most conserved residues are clustered into two distinct regions (one of .about.170 amino acids and the other of .about.260 amino acids), designated the X and Y regions, respectively. PLC.gamma..sub.1 also contains src-homology regions (SH2 and SH3) that appear to mediate the interaction between the enzyme and receptors with tyrosine kinase activity, such as the epidermal growth factor (EGF) receptor (Stahl et al. Nature, 332:269-272 (1988); Katan et al., Cell, 54:171-177 (1988)).
The PLC isozymes are activated by different mechanisms in response to stimulation of specific cell surface receptors. Coupling of PLC.delta. to specific receptors or downstream effectors has not been reported but this isozyme may be associated with mechanisms that regulate the tone of vascular smooth muscle. Activation of PLC.beta. is achieved by guanine nucleotide binding proteins of the Gq class.
To date, the cDNA for 6 distinct PI-PLC enzymes have been cloned. The enzymes range in size from 504 amino acids to 1250 amino acids, and are remarkably divergent considering that they exhibit similar biochemical properties. 4 of the 5 enzymes (PLC-.beta., PLC-.delta.1, PLC-.delta.2, and PLC-.gamma..sub.1 ) contain two domains approximately 250 amino acids in length which exhibit between 50 to 80% sequence similarity. The marked differences in DNA sequences for the different PI-PLC enzyme allows the selective targeting of one PI-PLC enzyme, without affecting other enzymes using antisense technology. The human cDNA clone has been reported for PLC-.delta.2, (Ohta et al., FEBS Lett., 242:31-35, 1988) and PLC-.gamma.1 (Burgess et al., Mol. Cell. Biol., 10:4770-4777 (1990)). The rest are rat cDNA clones. The genomic clones have not been reported for any of the PI-PLC enzymes.
All mammalian tissues which have been studied exhibit one or more PI-PLC enzymes. Generally, more than one enzyme exists in a single mammalian cell type. PI-PLC enzymes do exhibit tissue selectivity in their distribution. PLC-.beta. is found predominantly in neural tissues and is the major enzyme in the brain. PLC-.gamma..sub.1 is found in brain and many peripheral tissues. PLC-.delta..sub.2 is found in immune cells, and PLC-.delta..sub.1 appears to be predominantly in peripheral tissues. To date, a PI-PLC enzyme found exclusively in inflammatory cells has not been reported.
Point mutations of PLC-.delta.1 have been identified in the spontaneously hypertensive rat genome (Yagisawa et al., J. Hypertens. 9: 997-1004 (1991)). Biochemical studies have demonstrated the activation of PLC-.delta.1 (5 fold) in spontaneously hypertensive rats (Kato et al., J. Biol. Chem. 267:6483-6487 (1992)). The point mutations, situated in the putative catalytic domain, may be a major cause of the hypertension related phenomena of abnormal calcium homeostasis, a direct result of PLC-.delta.1 activation.
PI-PLC-.delta.2 appears to be an important enzyme in immunocompetent cells (Emori et al., J. Biol. Chem., 264:21885-21890). The protein is a moderately abundant protein comprising 0.1 to 0.05% of total cytosolic protein. No information is available concerning the genetic regulation of PI-PLC enzymes, mRNA or protein stability.
It has been established that a rapid synthesis of prostaglandins (PG) from arachidonic acid in macrophages usually accompanies inflammatory stimuli. Thus, inhibition of the release of arachidonic acid from macrophages would provide an effective control of PG synthesis and thereby inflammatory conditions. Recently, phospholiphase C has been characterized as an enzyme which is involved in the biosynthetic phosphatidylinositol-arachidonic acid-prostaglandin pathway. This finding is further substantiated by the observation that phospholipase C is inhibited by phenothiazine, a compound known to inhibit the stimulated release of arachidonic acid from macrophages and prostaglandins from platelets.
Activation of T cell antigen receptor (TCR/CD3) elicits a cascade of biochemical processes which are responsible for complex biological responses ranging from immune response to inflammation. The activation of PLC.gamma.1 can also be achieved through the action of nonreceptor protein tyrosine kinases in response to certain cell surface receptors in leukocytes (TCR) (Park et al., Proc. Natl. Acad. Sci. U.S.A. 88:5453-5456 (1991)). PLC.gamma.1 activation also occurs upon IgM ligation in B lymphocytes, IgE receptor (Fc.epsilon.RI) ligation in basophilic leukemia cells and IgG receptor (Fc.gamma.RI and Fc.gamma.RII) in monocytic cells (Liao et al., Proc. Natl. Acad. Sci. U.S.A. 89:3659-3663 (1992)). Thus, inhibition of PLC.gamma. activity, may be of therapeutic value in the treatment of inflammatory conditions.
PLC.gamma. is the only isozyme that is phosphorylated by activated tyrosine kinase growth factor receptors (Rotin et al., EMBO J., 11:559-567 (1992); Mohammadi et al., Mol. Cell. Biol., 11:5068-5078 (1992); Kim et al., Cell, 65:435-441 (1991)). Following growth factor stimulation, cytosolic PLC.gamma. is extensively and rapidly phosphorylated in vivo (50-70% of the PLC.gamma. molecules are modified within 5 minutes). This phosphorylation apparently induces the relocation of PLC.gamma. to the plasma membrane where presumably it is better able to interact with its phospholipid substrates. In vitro studies utilizing enzyme that had previously been immunoprecipitated from cells suggest that the catalytic activity of the phosphorylated form of PLC.gamma.1 is increased compared to that of the unphosphorylated form, although this effect also depends on the assay conditions. These results suggest that PLC.gamma. may be an important component of mitogenic signal transduction. Furthermore, altered PLC.gamma. activity may correlate with some disease states. For example, an increase in the concentration of PLC.gamma. has been documented in cells derived from primary human breast carcinomas which also overexpress the EGF receptor (Arteaga et al., Proc. Natl. Acad. Sci. U.S.A., 88, 10435-10439 (1991)). Thus, inhibition of PLC.gamma. activity, particularly of the activated form, may be of therapeutic value in the treatment of breast cancer.
In addition, PLC.gamma..sub.1 has been localized in vivo (immunohistochemistry) through many layers of the epidermis from a diverse series of hyperproliferative skin conditions, such as psoriasis, seborrheic keratosies and acrochordons (Nanney et al., Cell Growth and Differentiation, 3:233-239 (1992)). Thus inhibition of PLC.gamma. activity may be of therapeutic value in treating benign epidermal hyperplasia.
The recent demonstration that specific members of the Gq subfamily can activate different PLC-.beta. isozymes (e.g. Gq.alpha. activates PLC-.beta.1 ) (Smrcka et al., Science 25 1:804-807 (1991); Taylor et al., FEBS 286:214-216 (1991)) provides a connection of PLC-.beta. to a number of transmembrane signal transduction pathways. NIH3T3 cells transfected with an activated mutant of Gq.alpha. display a fully transformed phenotype, are highly tumorigenic in athymic nude mice (Kalinec et al., Mol. Cell Biol. 12:4687-4693 (1992)) and display greatly enhanced phospholipase C (PLC-.beta.) activity (DeVivo et al., J. Biol. Chem. 267:18263-18266 (1992)). Other mutatations in genes for the .alpha. subunits of some heterotrimeric G proteins (Lyons et al., Science 249:655-659 (1990); Vallar et al., Nature 330:556-558 (1987)), have been described and are associated with certain human endocrine tumours suggesting that activated G proteins may play a role in the oncogenic process. Thus, PLC-.beta. in addition to PLCT may be associated with human cancer.
Accordingly, it is an object of this invention to provide specific and selective inhibitors of phospholipase C which can be potent anti-inflammatory and analgesic agents useful in the treatment of inflammatory conditions, including rheumatoid arthritis, emphysema, bronchial inflammation, osteoarthritis, spondylitis, lupus, psoriasis, acute respiratory distress syndrome, gout, fever, and pain.
It is also the object of this invention to provide inhibitors of phospholipase C which are pharmaceutical agents useful in the treatment of certain forms of cancer, including breast cancer, and other hyperproliferative disease states of the epidermis.
Another object of this invention is to provide pharmaceutical compositions to be used in the administration of the novel phospholipase C inhibitors.
Still a further object of this invention is to provide a method of controlling and treating inflammation and pain by administering an effective amount of the compounds of the instant invention in a mammalian species in need of such treatment.